Our focus is the bacterial tad locus (tight adherence), a 14-gene cluster that directs synthesis of surface pili and is broadly conserved in Gram-negatives, Gram-positives, and Archaea. We discovered this locus in the Gram-negative pathogen Aggregatibacter actinomycetemcomitans, the causative agent of Localized Aggressive Periodontitis (LAP). Our prior studies have shown the tad locus is critical for A. actinomycetemcomitans surface adherence and pathogenicity. In this renewal application, we focus on two key proteins: Flp1, the pilin that mediates tight surface attachment, and TadZ, a tad-specific ATPase mimic that functions as a master localizer to recruit the Tad apparatus to the cell pole. These proteins are inviting therapeutic targets because they are either surface-accessible (Flp1) or have measurable molecular functions and interactions (TadZ). Our hypothesis is that functional perturbation of these proteins by genetic and chemical strategies will cause pilus defects that translate into loss of pathogenicity. We will pursue that hypothesis with three specific aims. First, we will manipulate the tad locus promoter in order to determine how overall tad expression levels affect pathogenicity. Second, we will define the TadZ properties that govern adherence and pathogenicity. Third, we will define Flp1 functional features that are required for pathogenicity. These studies will extend our current understanding of the A. actinomycetemcomitans tad locus function, based on in vitro assays, into critical tests of its biological function in colonization and virulence. In addition, our small-molecule screens may provide prospective lead compounds for development of new anti-infective agents, as well as molecular tools to facilitate inquiry into tad locus function in numerous other bacterial pathogens. PUBLIC HEALTH RELEVANCE: We study a set of genes that govern infection in numerous bacteria. Our current goals are to understand how two key genes work to control infection, and to find chemicals that block their activity. These chemicals may be developed into new antibiotic drugs, supporting therapy for multiple types of infection.